Project Summary and Relevance Since the proposal of the nucleosome hypothesis, more than 30 years ago, an extensive body of work has demonstrated the critical role of chromatin in gene regulation. In vitro and in vivo experiments from different model systems have unequivocally shown that covalent histone modifications provide a signal for the recruitment of chromatin modifying proteins that activate or silence gene expression. High throughput approaches recognized that these covalent marks have different genomic distribution, some are enriched on promoter sequences, others mark enhancers, whereas a few decorate the transcriptionally inactive and repetitive portion of our genome. Moreover, genetic and pharmacological experiments showed that these modifications, despite being termed epigenetic, appear extremely dynamic; the enzymes that apply them are in constant competition with the enzymes that erase them. Therefore, each nucleus and even each DNA locus adopts distinct epigenetic states in space and time. The dynamic nature of the epigenome poses a significant challenge towards the understanding of the exact order of events that culminate in gene expression or silencing during development and differentiation. Overcoming this challenge becomes even more critical by the realization that the epigenome has a profound effect in human health and that perturbations thereof result in disease. Therefore, new technologies need to be developed for the monitoring of the epigenome in a single cell resolution in living cells. Here, we propose the use of the powerful bimolecular Fluorescence Complementation technology towards the live imaging of the mammalian epigenome. Our experiments aim to visualize the extend, and nuclear distribution of different histone marks in differentiating cells and to assign the position of these marks in relation to specifically labeled DNA loci. Moreover, we will use a novel imaging system, soft X-ray tomography that combines remarkable spatial resolution with the benefits of fluorescent microscopy. With this technology we will gain structural insight of different epigenetic domains in the fully hydrated mammalian nucleus. The information generated by the combination of these innovative approaches will allow a better understanding of epigenetic regulation in model organisms and will promote the development of therapeutic epigenetic compounds. Moreover, these experiments will lay the foundation of an epigenomic analysis of the genetically intractable human being and will provide sensitive tools for the future for the diagnosis or even prognosis of diseases linked to epigenetic perturbations. PUBLIC HEALTH RELEVANCE: Project Narrative The epigenetic state of a human cell has profound consequences in the transcriptional program of this cell, the proper function of the organ at which the cell belongs to, and the overall health of the human being. Therefore, our proposed technologies, which will allow for the live imaging of the epigenetic regulation in vivo, will assist the better understanding of epigenetic processes and how these result in disease, will allow the development of novel therapeutic compounds and will provide powerful future diagnostic tools for epigenetic diseases.